Bcr-Abl Kinase Inhibitor Research Articles

Endothelium as a Source of Cardiovascular Toxicity From Antitumor Kinase Inhibitors.

Kinase inhibitors (KIs) targeting oncogenic molecular pathways have revolutionized cancer therapy. By directly targeting specific tumor-driving kinases, targeted therapies have fewer side effects compared with chemotherapy. Despite the enhanced specificity, cardiovascular side effects have emerged with many targeted cancer therapies that limit long-term outcomes in patients with cancer. Endothelial cells lining all blood vessels are critical to cardiovascular health and are also exposed to circulating levels of systemic anticancer therapies. Both on- and off-target perturbation of signaling pathways from KIs can cause endothelial dysfunction, resulting in cardiovascular toxicity. As such, the endothelium is a potential source, and also a therapeutic target for prevention, of cardiovascular toxicity. In this review, we examine the evidence for KI-induced endothelial cell dysfunction as a mechanism for the cardiovascular toxicities of vascular endothelial growth factor inhibitors, BCR-Abl (breakpoint cluster region-Abelson proto-oncogene) KIs, Bruton tyrosine inhibitors, and emerging information regarding endothelial toxicity of newer classes of KIs.

Rationally designed BCR-ABL kinase inhibitors for improved leukemia treatment via covalent and pro-/dual-drug targeting strategies

BackgroundChronic Myeloid Leukemia (CML) is a blood cancer that remains challenging to cure due to drug resistance and side effects from current BCR-ABL inhibitors. There is an urgent need for novel and more effective BCR-ABL targeting inhibitors and therapeutic strategies to combat this deadly disease. MethodWe disclose an “OH-implant” strategy to improve a noncovalent BCR-ABL inhibitor, PPY-A, by adding a hydroxyl group to its scaffold. By taking advantage of this OH “hot spot”, we designed a panel of irreversible covalent kinase inhibitors and hypoxia-responsive pro-/dual-drugs, and their biological activities were studied in vitro, in cellulo and in vivo. ResultThe resulting compound B1 showed enhanced solubility and biological activity. B4 achieved sustained BCR-ABL inhibition by forming a stable covalent bond with ABL kinase. Hypoxia-responsive prodrug P1 and dual-drugs D1/D2/D3 demonstrated significant anti-tumor effects under hypoxic conditions. The in vivo studies using K562-xenografted mice showed that B1 displayed superior antitumor activity than PPY-A, while P1 and D3 offered better safety profiles alongside significant tumor control. ConclusionWe have successfully developed a chemical biology approach to convert a known noncovalent BCR-ABL inhibitor into more potent and safer inhibitors through covalent and pro-/dual-drug targeting strategies. Our “OH-implant” approach and the resulting drug design strategies have general applicability and hold promise for improvement the performance of various other reported drugs/drug candidates, thereby providing advanced medicines for disease treatment.

The Progress of Small Molecule Targeting BCR-ABL in the Treatment of Chronic Myeloid Leukemia.

Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease. According to the American Cancer Society's 2021 cancer data report, new cases of CML account for about 15% of all leukemias. CML is generally divided into three stages: chronic phase, accelerated phase, and blast phase. Nearly 90% of patients are diagnosed as a chronic phase. Allogeneic stem cell transplantation and chemotherapeutic drugs, such as interferon IFN-α were used as the earliest treatments for CML. However, they could generate obvious side effects, and scientists had to seek new treatments for CML. A new era of targeted therapy for CML began with the introduction of imatinib, the first-generation BCR-ABL kinase inhibitor. However, the ensuing drug resistance and mutant strains led by T315I limited the further use of imatinib. With the continuous advancement of research, tyrosine kinase inhibitors (TKI) and BCR-ABL protein degraders with novel structures and therapeutic mechanisms have been discovered. From biological macromolecules to classical target protein inhibitors, a growing number of compounds are being developed to treat chronic myelogenous leukemia. In this review, we focus on summarizing the current situation of a series of candidate small-molecule drugs in CML therapy, including TKIs and BCR-ABL protein degrader. The examples provided herein describe the pharmacology activity of small-molecule drugs. These drugs will provide new enlightenment for future treatment directions.

Population Pharmacokinetics and Pharmacogenetics Analyses of Dasatinib in Chinese Patients with Chronic Myeloid Leukemia.

Dasatinib, a second-generation tyrosine kinase inhibitor of BCR-ABL 1, used for first-line treatment of Philadelphia chromosome-positive chronic myeloid leukemia (CML), exhibits high pharmacokinetic (PK) variability. However, its PK data in Chinese patients with CML remains rarely reported to date. Thus, we developed a population pharmacokinetic (PPK) model of dasatinib in Chinese patients and identified the covariate that could explain the individual variability of PK for optimal individual administration. PPK modeling for dasatinib was performed based on 754 plasma concentrations obtained from 140 CML patients and analysis of various genetic and physicochemical parameters. Modeling was performed with nonlinear mixed-effects (NLME) using Phoenix NLME. The finally developed model was evaluated using internal and external validation. Monte Carlo simulations were used to predict drug exposures at a steady state for various dosages. The PK of dasatinib were well described by a two-compartment with a log-additive residual error model. Patients in the current study had a relatively low estimate of CL/F (126 L/h). A significant association was found between the covariate of age and CL/F of dasatinib, which was incorporated into the final model. None of the genetic factors was confirmed as a significant covariate for dasatinib. The results of external validation with 140 samples from 36 patients were acceptable. Simulation results showed significantly higher exposures in elderly patients. This study's findings suggested that low-dose dasatinib would be better suited for Chinese patients, and the dosage can be appropriately reduced according to the increase of age, especially for the elderly.

A thin-film molecularly imprinted polymer for the measurement of tyrosine kinase inhibitors in human plasma

Tyrosine kinase inhibitors (TKIs) are a class of drugs that have found broad application in cancer therapies. These include imatinib, the first TKI used as an inhibitor of BCR-ABL kinase to treat chronic myelogenous leukemia, as well as second generation (e.g., nilotinib and dasatinib) and third generation (e.g., ponatinib) TKIs with increased potency, specificity, and binding site affinity specificity. Although the evolution of TKIs have brought dramatic increases to life expectancy, adverse events are likely due to the relatively high dosage required for treatment and the high pharmacokinetic variability between individuals. Therapeutic drug monitoring can be employed to fine tune the dosage for better control of the treatment. In this work, we present an extraction system that incorporates a novel molecularly imprinted polymer (MIP) capable of the rapid extraction of four TKIs: imatinib, nilotinib, dasatinib, and ponatinib from human plasma. The system is comprised of 96 thin-film microextraction devices in a standard flat-bottomed 96-well plate, which allows for rapid and reproducible concurrent TKIs extraction with determination by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Extraction, washing, and desorption protocols are simplified, with minimal manual handling and can be fully automated. Inter-device and batch-to-batch variability is low (∼5%), commending these for single-use applications. The linear ranges for all four TKIs are broad, with limits of detection and quantification far below the lowest clinically significant values and upper limits above the therapeutic ranges. Overall, these devices would be suitable for clinical applications or therapeutic drug monitoring of dasatinib, imatinib, nilotinib, and ponatinib, either individually or in any combination.

BAX as the mediator of C-MYC sensitizes acute lymphoblastic leukemia to TLR9 agonists

BackgroundThe prognosis of B-cell acute lymphoblastic leukemia (B-ALL) has improved significantly with current first-line therapy, although the recurrence of B-ALL is still a problem. Toll-like receptor 9 (TLR9) agonists have shown good safety and efficiency as immune adjuvants. Apart from their immune regulatory effect, the direct effect of TLR9 agonists on cancer cells with TLR9 expression cannot be ignored. However, the direct effect of TLR9 agonists on B-ALL remains unknown.MethodsWe discussed the relationship between TLR9 expression and the clinical characteristics of B-ALL and explored whether CpG 685 exerts direct apoptotic effect on B-ALL without inhibiting normal B-cell function. By using western blot, co-immunoprecipitation, immunofluorescence co-localization, and chromatin immunoprecipitation, we explored the mechanism of the apoptosis-inducing effect of CpG 685 in treating B-ALL cells. By exploring the mechanism of CpG 685 on B-ALL, the predictive biomarkers of the efficacy of CpG 685 in treating B-ALL were explored. These efficiencies were also confirmed in mouse model as well as clinical samples.ResultsHigh expression of TLR9 in B-ALL patients showed good prognosis. C-MYC-induced BAX activation was the key to the effect of CpG oligodeoxynucleotides against B-ALL. C-MYC overexpression promoted P53 stabilization, enhanced Bcl-2 associated X-protein (BAX) activation, and mediated transcription of the BAX gene. Moreover, combination therapy using CpG 685 and imatinib, a BCR-ABL kinase inhibitor, could reverse resistance to CpG 685 or imatinib alone by promoting BAX activation and overcoming BCR-ABL1-independent PI3K/AKT activation.ConclusionTLR9 is not only a prognostic biomarker but also a potential target for B-ALL therapy. CpG 685 monotherapy might be applicable to Ph− B-ALL patients with C-MYC overexpression and without BAX deletion. CpG 685 may also serve as an effective combinational therapy against Ph+ B-ALL.

Development of optimized pyrimido-thiazole scaffold derivatives as anticancer and multitargeting tyrosine kinase inhibitors using computational studies

Cancer is one of the frontier causes of leading death ratesglobally and one of the most researched domains. Despite many drugs available in the market currently, their effectiveness towards resistance, randomly mutating cancer targets, and side effects initiate the necessity of developing target selective and effective anti-cancer drugs.This study aimed to develop a congeneric series of fused pyrimidine and thiazole-containing hybrid series and evaluate its anticancer potential using in-silico tools. Molecular modeling strategies were used to develop various derivatives of fused thiazole-pyrimidines as anticancer agents for target-based drug discovery. Strategically, based on Structural Activity Relationship (SAR), of the synthesized or studied compounds, a combinatorial library was generated using the lead grow tool to develop a possible combination of various derivatives. Using Swiss ADME and Osiris; the ADMETproperties were predicted and screened molecules were docked using Auto-dock vina and Swiss Dockto obtain possible interactions with 5 listed targets of Tyrosine Kinase Inhibitors (TKI's) namely BCR-ABL kinase (PDB- 2GQG), c-ABL Kinase (PDB-3PYY), EGFR(PDB-1M17), VEGFR(PDB- 4ASD), and BRAF inhibitors (PDB- 2FB8). SAR of the pyrimido-thiazole molecules was derived from the literature survey and substitutions that enhance the anticancer activity were incorporated into the Combilib to generate a library of 221 compounds. This library of compounds was subjected to a Lipinski Filter. About 45 compounds were screened and tested for their drug-likeliness parameters. The 24 compounds that qualified with good absorption, drug-likeliness, and ADME were tested for their toxicity for developing relatively safe drugs with low side effects. These molecules were docked intofivedifferent PDB's and their binding affinity scores along with prominent interactions were noted. Compound 33 has shown comparable binding affinity and key interactions with all the five PDB's and thus can be used as a lead for further investigations. These studies have helped to optimize the relationship between fused pyrimidine and thiazoles as anticancer Tyrosine Kinase Inhibitors.

Rxra Overexpression Improves the Sensitivity to Imatinib In-Vitro/in-Vivo By Disrupting the Oxidative Capacity of Chronic Myeloid Leukemia Cells

Most patients who receive BCR-ABL1 kinase inhibitor therapy for CML see profound molecular responses. However, due to drug resistance clones and leukemic stem cells, not all patients benefit from TKI therapy. We have previously reported the dysregulated expression of nuclear hormone receptors in CML cell lines based on the sensitivity to Imatinib (IM) (Karathedath et al., ASH annual meeting abstracts 2013). We observed Retinoid X receptor alpha (RXRA) expression to be markedly down-regulated in CML cell lines (KCL22, LAMA84) that were inherently resistant to IM compared to IM-sensitive cell lines (EM2, KU812). Here, we undertook a series of studies to decipher the role of RXRA in mediating sensitivity to IM. Initially, we stably overexpressed RXRA in the LAMA84 cell line with the lowest expression of RXRA. Post-RXRA overexpression (OE), we observed a significant reduction in cell proliferation in a time-dependent manner compared to empty vector (EV) transduced cells as assessed by the trypan blue exclusion (35.7 Vs. 25.9 hours). Next, we evaluated the sensitivity to IM by apoptosis assay, and RXRA OE cells were significantly sensitized to IM (58% vs. 21%. P<0.01). We then assessed the sensitivity to other TKIs nilotinib and dasatinib in the RXRA OE cells and found increased sensitivity. We evaluated the expression of major pro-survival down streaming kinase signaling pathways of BCR-ABL (AKT, CRKL, and STAT5) in the RXRA OE cells post IM exposure. Interestingly, we observed diminished expression of p-CRKL in RXRA OE cells compared to EV, and further treatment with IM completely abolished the expression of p-AKT, p-STAT5, and p-CRKL in the RXRA OE cells vs. EV. It is well established that the leukemic cells show a metabolic shift towards oxidative phosphorylation to elicit therapy resistance; therefore, we performed extracellular flux analysis (Seahorse) to identify metabolic alteration post-RXRA OE. Interestingly, RXRA OE caused a significant reduction in the basal OCR rates, spare respiratory capacity, and ATP production compared to EV. We also measured the mitochondrial membrane potential (MMP) using JC-1 dye and found that reduced MMP in RXRA OE cells compared to EV. This suggests RXRA OE reduced the oxidative capacity of the LAMA84 cell line and thereby increased the sensitivity to TKI. We performed xenograft transplantation of the RXRA OE/EV cells and assessed the engraftment capacity and sensitivity to IM in-vivo. RXRA OE leads to reduced engraftment in the bone marrow and improved survival compared to EV transplanted mice (Median survival 50 vs 37 days). Further RXRA OE also led to an increased response to IM in-vivo, which resulted in better survival than IM-treated EV mice (Median survival 57 vs 48 days). We probed for RXRA expression between CML CD34+ cells and health donor CD34+ cells and found significantly reduced expression. Next we assessed the effect of clinically available ligands specific to RXRA (Acitretin,Bexarotene and 9-cis-Retinoic acid) on CML CD34+ cells in combination with IM. Intriguingly, pretreament of CML CD34+cells with RXRA ligands followed by IM treatment, markedly reduced the viability and colongenic capacity of these cells compared to IM treatment alone. In conclusion, we have identified that enforced expression of RXRA in CML cells improved sensitivity to TKI in-vitro and in-vivo. The profound sensitivity to TKI was found to be RXRA's disruptive effect on the OXPHOS metabolism. Interestingly RXRA OE reduced the engraftment potential of CML cells in-vivo, indicating a mechanism for altering the stemness capacity of CML cells. Further work is ongoing to elucidate the molecular mechanisms orchestrated by RXRA in CML cells.

Abstract 10554: Major Cardiac Side Effects of BTK and BCR-ABL Kinase Inhibitor Therapy in Cancer Patients: A Global WHO Database Analysis

Introduction: Bruton’s tyrosine kinase (BTK) and BCR-ABL kinase inhibitors have transformed the treatment for hematological cancers, such as mantle cell lymphoma and chronic myelogenous leukemia. However, these therapies present unique cardiac events that remain underexplored. Hypothesis: We hypothesize that the two groups of targeted therapies commonly used to treat hematological cancers will differ in cardiac side-effect profiles. Methods: The World Health Organization database, VigiBase ®, contains over 20 million Individual Case Safety Reports and is maintained by the Uppsala Monitoring Centre. Reports for unique cardiac events associated with the use of BTK and BCR-ABL kinase inhibitors were analyzed. Reporting odds ratios (ROR), 95% confidence intervals (CI) and chi-squared tests were used to compare differences in cardiac side-effect profiles between the two groups of therapies. Results: A total of 24,385 adverse cardiac events from the use of BTK inhibitors (ibrutinib, acalabrutinib, zanubrutinib, tirabrutinib) and BCR-ABL kinase inhibitors (imatinib, dasatinib, nilotinib, bosutinib, ponatinib) have been observed based on records obtained to-date. Compared to BCR-ABL kinase inhibitors, BTK inhibitors have significantly increased reporting for atrial fibrillation (ROR: 6.995; 95% CI: 6.493-7.537; P &lt; 0.001) and ventricular tachycardia (ROR: 1.829; 95% CI: 1.342-2.493; P &lt; 0.001). However, BCR-ABL inhibitors have significantly increased reporting for myocardial infarction (ROR: 2.077; 95% CI: 1.863-2.316; P &lt; 0.001) but similar reporting for congestive cardiac failure (ROR: 0.949; 95% CI: 0.852-1.058; P = 0.343). The median onset of ventricular tachycardia from the use of BTK inhibitors is 176 days (interquartile range: 48-636 days). For BCR-ABL kinase inhibitors, the median onset of ventricular tachycardia is similar at 137 days (interquartile range: 92-396 days), P = 0.741 from nonparametric tests. Conclusion: We conclude that BTK inhibitors are associated with arrhythmic events, while BCR-ABL kinase inhibitors are associated with myocardial infarction. Both groups have similar reporting for congestive cardiac failure. Research is underway to study the mechanisms behind these novel side-effect profiles.

Cardiotoxicity of anticancer drugs and radiotherapy in patients with hematologic malignancies and solid tumors

Cardioncology has emerged as a new field at the intersection of cardiology and oncology. Despite the fact that improving efficiency of antitumor treatment increased the survival of oncological hematological patients, the long-term cardiovascular consequences of this treatment have become more clinically significant.Despite the effectiveness of modern methods of treatment, some drugs, such as Bcr-Abl kinase inhibitors, anthracyclines, HER2/Erbb2 inhibitors, vascular endothelial growth factor inhibitors, fluoropyrimidines, as well as radiation therapy can have a pronounced effect on the cardiovascular system. These toxic effects lead to cardiac arrhythmia, heart failure, vascular toxicity and even death. It is important for hematologists, oncologists and cardiologists to understand the basic diagnostic and treatment strategies that should be used in the event of toxicity of this kind. At a time when, due to the developed cardiotoxicity, antitumor therapy should be discontinued, in some cases, it is possible to consider continuing treatment with caution and careful monitoring.

Safety, pharmacodynamic, and clinical response evaluation of nilotinib and paclitaxel in adults with refractory solid tumors.

3026 Background: The combination of the BCR-Abl kinase inhibitor nilotinib and the anti-tubulin agent paclitaxel was identified in the NCI-ALMANAC study to have greater-than-additive activity in the NCI-60 cell line panel and greater-than-single-agent antitumor activity in xenograft models, in which this combination induces tumor epithelial-mesenchymal transition (EMT). A phase 1 study was initiated to establish the safety, tolerability, and recommended phase 2 dose (RP2D) of this combination in patients (pts) with advanced solid tumors and to examine the pharmacokinetic (PK) and pharmacodynamic (PD) effects of the combination to understand the mechanism of action (NCT02379416). The dose escalation phase established the RP2D as 300 mg oral nilotinib twice daily and 80 mg/m2 intravenous paclitaxel on days (D) 1, 8, and 15 of each 28-day cycle. Here, we report the safety, preliminary PD, and efficacy data for this combination. Methods: Nilotinib and paclitaxel were administered as noted above, with a 1-day (escalation cohort) or 2-day (expansion cohort) paclitaxel-only run-in during the first cycle to enable comparison of the PK and PD effects of the combination vs. single-agent paclitaxel. Paired biopsies to assess tumor molecular response were collected from expansion cohort pts at baseline, cycle (C) 1 D2, and C1D28, with an optional biopsy at progression; accrual continued until ≥ 12 sufficient-quality paired biopsies were obtained. Blood specimens to assess molecular responses in circulating tumor cells (CTCs) were obtained at several timepoints during C1 and longitudinally every cycle thereafter. EMT biomarkers were measured in tumor and CTC specimens using quantitative immunofluorescence microscopy assays. Results: A total of 44 pts were enrolled. Three pts had partial responses (PR), and 1 had an unconfirmed PR (9%); 23 pts (52%) had a best response of stable disease (SD), including 7 pts on study for ≥ 10 cycles. The most common grade (Gr) 3-4 treatment-related adverse events were hematologic and hypophosphatemia. No pts experienced Gr ≥ 3 peripheral neuropathy. The median time on treatment was 67 days. Two pts with granulosa cell ovarian carcinoma had durable responses, completing 74+ and 64 cycles. Multiple patient biopsies and corresponding CTC specimens exhibited treatment-induced EMT. Longitudinal analysis of CTC EMT phenotypes in the 2 pts with extended PR revealed a substantial increase in mesenchymal-like CTCs prior to progression for the pt on study for 64 cycles; such increases were not observed in the pt still on study after 74+ cycles. Further PD analyses are ongoing. Conclusions: The combination of nilotinib and paclitaxel demonstrates promising disease control with durable response in select patients. Tumor PD analyses to discover the underlying pharmacology of this active regimen are ongoing. Funded by NCI Contract No. HHSN261201500003I. Clinical trial information: NCT02379416.

Design, synthesis, and biological evaluation of trizole-based heteroaromatic derivatives as Bcr-Abl kinase inhibitors

Bcr-Abl is a key driver in the pathophysiology of CML. Broadening the chemical diversity of Bcr-Abl kinase inhibitors to overcome drug resistance is a current medical demand for CML treatment. As a continuation to our research, a series of compounds with heteroaromatics-trizole scaffold as hinge binding moiety (HBM) were developed as Bcr-Abl inhibitors based on in silico modeling analysis. Biological results indicated that these compounds exhibited a significantly enhanced inhibition against Bcr-AblWT and Bcr-AblT315I in kinases assays, along with improved anti-proliferative activities in leukemia cell assays, compared with previous disclosed compounds. In particular, compounds 9f, 28c, 31, and 44c displayed comparable even better potency with that of Imatinib in enzymatic assay and cell assays including K562 cells and adriamycin-resistant K562/A cells. Moreover, compounds 9f, 28c, and 44c exhibited potent inhibition activities against K562R cells bearing T315I mutant with IC50 of 13.35 μM, 40.14 μM, and 1.91 μM, respectively, outperforming that of Imatinib. Meanwhile, the inhibition of Bcr-Abl activity in Ba/F3 cells demonstrated that these compounds exerted effects mainly by acting on Bcr-Abl. Additionally, compounds 9f, 28c, and 44c effectively induced apoptosis, arrest the cell cycle at S or G2/M phase, and inhibited phosphorylation of Bcr-Abl and STAT5 in a dose-dependent manner. Docking studies indicated that trizole indeed retained the hydrophobic interaction of aromatic heterocycles with hinge region, and ADME prediction suggested that tested compounds had a favorable safety profile. Therefore, aromatic heterocycles incorporated with trizole could serve as a promising HBM for Bcr-Abl inhibitors with proline as fexibile linker, and compounds 9f, 28c, especially 44c could be served as a starting point for further optimization.

Bcr-Abl drives the formation of Hsp70/Bim PPI to stabilize oncogenic clients and prevent cells from undergoing apoptosis

Although tyrosine kinase inhibitors that inhibit Bcr-Abl kinase activity have shown excellent efficacy in the clinical application of CML patients, it still a challenge to discover alternative targets and novel therapies because of the emergence of Bcr-Abl-independent resistance. Most recently, Hsp70/Bim complex was revealed that driven by Bcr-Abl and testified as a specific target for CML because it folds and stabilizes many Hsp70 oncogenic substrates that mediate CML specific signaling pathways. However, the relationship between Bcr-Abl and Hsp70/Bim complex and how the chaperone complex contributes to Bcr-Abl-driven leukemogenic cells remain unclear. Herein, with the help of S1g-2, a specific small-molecule inhibitor of Hsp70/Bim complex, and Bcr-Abl knockdown to induce a panel of cancer cell lines apoptosis, we illustrated that Bcr-Abl could prevent cells from undergoing apoptosis mainly by driving the formation of Hsp70/Bim complex both in Bcr-Abl positive CML cells and ALL cells. Through cell-based Co-immunoprecipitation experiments, we identified that Bcr-Abl could stabilize oncogenic clients including AKT and eIF4E mainly by driving the formation of Hsp70/Bim complex in Bcr-Abl positive cells. Moreover, we identified that Bim mediates interactions of Hsp70 and Bak in Bcr-Abl positive cells. Together, the target identification of Hsp70/Bim complex could make it as a promising anticancer target for Bcr-Abl positive leukemia treatment.

Imatinib can act as an Allosteric Activator of Abl Kinase

Imatinib is an ATP-competitive inhibitor of Bcr-Abl kinase and the first drug approved for chronic myelogenous leukemia (CML) treatment. Here we show that imatinib binds to a secondary, allosteric site located in the myristoyl pocket of Abl to function as an activator of the kinase activity. Abl transitions between an assembled, inhibited state and an extended, activated state. The equilibrium is regulated by the conformation of the αΙ helix, which is located nearby the allosteric pocket. Imatinib binding to the allosteric pocket elicits an αΙ helix conformation that is not compatible with the assembled state, thereby promoting the extended state and stimulating the kinase activity. Although in wild-type Abl the catalytic pocket has a much higher affinity for imatinib than the allosteric pocket does, the two binding affinities are comparable in Abl variants carrying imatinib-resistant mutations in the catalytic site. A previously isolated imatinib-resistant mutation in patients appears to be mediating its function by increasing the affinity of imatinib for the allosteric pocket, providing a hitherto unknown mechanism of drug resistance. Our results highlight the benefit of combining imatinib with allosteric inhibitors to maximize their inhibitory effect on Bcr-Abl.

Mutation in Abl kinase with altered drug-binding kinetics indicates a novel mechanism of imatinib resistance

Protein kinase inhibitors are potent anticancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for chronic myeloid leukemia by 80%, but 22 to 41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, where more than a hundred different mutations have been identified. Some mutations are located near the imatinib-binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug-binding site, and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies. Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease relevant or resistance causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than twofold compared to Abl wild type. Surprisingly, one-third of mutations in the Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified three clinical Abl mutations that bind imatinib with wild type-like affinity but dissociate from imatinib considerably faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the nonequilibrium environment of the body where drug export and clearance play critical roles.

Flavonoid Derivatives Targeting BCR-ABL Kinase: Semisynthesis, Molecular Dynamic Simulations and Enzymatic Inhibition.

Natural products have been universally approached in the research of novel trends useful to detail the essential paths of the life sciences and as a strategy for pharmacotherapeutics. This work focuses on further modification to the 6-hydroxy-flavanone building block aiming to obtain improved BCR-ABL kinase inhibitors. Ether derivatives were obtained from Williamson synthesis and triazole from Microwave- assisted click reaction. Chemical structures were finely characterized through IR, 1H and 13C NMR and HRMS. They were tested for their inhibitory activity against BCR-ABL kinase. Two inhibitors bearing a triazole ring as a pharmacophoric bridge demonstrated the strongest kinase inhibition at IC50 value of 364 nM (compound 3j) and 275 nM (compound 3k). 6-hydroxy-flavanone skeleton can be considered as a promising core for BCR-ABL kinase inhibitors.

Abstract 1463: ATP-site inhibitor olverembatinib, HQP1351, enhanced the effect of allosteric inhibitor on the resistance conferred by the compound mutations of BCR-ABL

Abstract Treatment with tyrosine kinase inhibitors (TKIs) directed against the ATP-binding site of BCR-ABL promotes recovery of Ph+ leukemia. However, emergence of gatekeeper mutation T315I and compound mutants confer resistance to these TKIs. The allosteric inhibitor, asciminib, effectively inhibits BCR-ABL kinase through binding to the myristoyl-binding site. Combining asciminib with ponatinib can overcome only a subset of the resistance caused by BCR-ABL compound mutants. Olverembatinib (HQP1351) is a new generation TKI targeting BCR-ABL and currently in development for r/r CML. The purpose of this study is to evaluate whether a novel combination of olverembatinib and asciminib, targeting both ATP pocket and allosteric region of BCR-ABL protein, can promote the inhibitory effect on the kinase harboring compound mutations. A series of cell lines expressing BCR-ABL single or compound mutations were constructed based on BaF3 murine pro-B cell line. Effect of olverembatinib as a single agent or in combination with asciminib were analyzed using antiproliferation assay, Western blot and FACS assays in vitro. In vivo efficacy was evaluated using the syngeneic mouse model derived from BaF3 cells with T315I or compound mutations. The cell based antiproliferation studies demonstrated superior activity of olverembatinib toward BCR-ABL single or compound mutations with IC50 values ranging between 6-300 nM. In particular, olverembatinib was more effective than ponatinib, asciminib and other TKIs against those compound mutations. Moreover, the combination of olverembatinib with asciminib was highly effective against BCR-ABL compound mutations, especially those containing T315I. In vivo studies further revealed that co-administration of olverembatinib with asciminib resulted in significant prolongation of survival compared to single agents. Importantly, the anti-tumor effect was more potent than that of ponatinib plus asciminib in models harboring compound mutations. In terms of mechanism, the combined treatment synergistically downregulated phosphorylation of BCR-ABL and the downstream proteins CRKL and STAT5, and augmented cleavage of Caspase-3 and PARP-1, thus triggered apoptosis and subsequently enhanced the antitumor effects. Our results demonstrated that the combination of ATP binding site inhibitor olverembatinib and allosteric inhibitor have synergistic anti-tumor effect on tumor cells harboring single or compound mutations in BCR-ABL. This novel strategy may help to overcome the secondary compound mutations post the treatment with TKIs. Citation Format: Guangfeng Wang, Jing Lv, Chunyang Tang, Ping Min, Li Rui, Fei Zhang, Lvcheng Wang, Yangfeng Ge, Kejie Lian, Eric Liang, Dajun Yang, Yifan Zhai. ATP-site inhibitor olverembatinib, HQP1351, enhanced the effect of allosteric inhibitor on the resistance conferred by the compound mutations of BCR-ABL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1463.

Allosteric Bcr-Abl Inhibitor ABL001 Overcomes Bcr-Abl Tyrosine Kinase Inhibitor Resistance and Enhances MDM2 Inhibitor CGM097 Activity in Blast Crisis CML Cells

Despite Bcr-Abl tyrosine kinase inhibitors (TKIs) are highly effective in CML, development of TKI-resistance leading to blast crisis (BC) progression is still a challenge in CML therapy. This has motivated the generation of novel agents and combination strategies to overcome the resistance. ABL001 is a small molecule allosteric Bcr-Abl kinase inhibitor that occupies the myristoyl pocket, leading to a kinase autoinhibition conformation. ABL001 has entered phase I clinical trials to treat patients with chronic, accelerated, or blast phase CML that relapsed from TKIs (Hughes et al., ASH 2016). Its activity in BC CML and ability to enhance the traditional TKIs that target ATP binding site are under investigation. TP53, a key tumor suppressor gene plays a crucial role in mediating cell death in part through regulating the expression or activity of Bcl-2 family proteins. Although highly mutated in solid tumors, the mutation rate of TP53 is relatively low in CML. However, p53 antagonist, MDM2 can be regulated by Bcr-Abl signaling and ARF, which inhibits MDM2 is frequently deleted in BC CML suggesting therapeutic potentials of inhibition of MDM2 in BC CML. Indeed, activation of p53 by MDM2 inhibition has been shown to enhance the toxicity of TKIs in BC CML (Carter et al., Oncotarget 2015, 6(31):30487). In this study, we examined the activity of ABL001 in CML cells and investigated if ABL001 enhances the activity of TKIs and synergizes with MDM2 inhibitor CGM097 in samples from heavily treated TKI resistant BC CML patients.BaF3 cells expressing BCR-ABL with or without mutations, human CML cell lines (KBM5 and KBM5-STI-T315I), and peripheral blood mononuclear cells (PBMC) isolated from primary BC CML patient samples were treated with various doses of ABL001, CGM097 or Nilotinib alone or in combination. Apoptosis was determined by Annexin V/7-AAD staining in cell lines and CD45+ (bulk), CD34+CD38+ and CD34+CD38- cells of CML patient PBMC samples. EC50 was calculated using CompuSyn or Graphpad. Protein levels were examined by Western blotting.We found that ABL001 induced apoptosis in cell lines expressing BCR-ABL (BaF3p210 and KBM5) or mutant BCR-ABL (BaF3p210E255K and KBM5-STI-T315I) but had limited activity in BaF3p210T315I cells. Importantly, ABL001 induced apoptosis in bulk, CD34+CD38+, or/and CD34+CD38- cell populations at low nM concentrations in 5 out of 6 BC CML patient samples tested. Patient samples that responded to ABL001 had either T315I and/or E255 mutations in BCR-ABL kinase domain. ABL001 inhibited Bcr-Abl signaling as measured by the levels of p-Bcr-Abl/p-Stat5/p-Crkl in BCR-ABL positive cells with or without BCR-ABL mutations. ABL001 showed higher activity than Nilotinib in 4 out of 5 responding patient samples. Nilotinib did not significantly enhance ABL001-induced apoptosis in the tested BC CML patient samples and CML cell lines. Inhibition of MDM2 with CGM097 activated p53 and induced apoptosis in BC CML patient samples. The activity of ABL001 was enhanced by CGM097 in the majority of patient samples which did not have p53 mutations. ABL001 combined with CGM097 was more effective than the combination of nilotinib with CGM097. The combinations of TKI and CGM097 were more effective in killing leukemia cells than each agent alone even when leukemia cells were co-cultured with MSCs. Neither ABL001 or CGM097 alone, nor the combination showed toxicity on normal bone marrow stem/progenitor cells.In conclusion, our results demonstrate that the allosteric Bcr-Abl Inhibitor ABL001 overcomes TKI-resistance and enhances MDM2 inhibitor CGM097 activity in BC CML cells. The combination of ABL001 and CGM097 warrants clinical evaluation. DisclosuresCarter:novartis: Research Funding; Daiichi Sankyo: Research Funding.

Evaluating the Role of Lower Dose Dasatinib in Newly Diagnosed Early Chronic Phase-Chronic Myeloid Leukemia (CML-CP)

Introduction: Dasatinib is a potent tyrosine kinase inhibitor (TKI) of BCR-ABL . It is currently approved for the treatment of all phases of CML. Approved dose in the chronic phase is 100mg daily. This is however associated with notable side effects mainly myelosuppression and pleural effusion, leading to dose reductions/interruptions. Lower doses of dasatinib have been shown to be effective in elderly CML patients.Objective: To evaluate the efficacy and toxicity profile of lower dose dasatinib, at 50 mg orally daily in patients with early CML-CP.Methods: All patients presenting to our institution in early CML-CP were eligible to participate. Prior TKI therapy for up to 1 month was allowed. Responses were assessed according to the European LeukemiaNet guidelines (Baccarani et al. Blood 2013 122.872:884).Results: From March 2016 to July 2017, 56 patients have been enrolled. Median age is 46 years (range 22-80). Patients categorized by Sokal risk are: low 34; intermediate 16 and high 6. Nine patients received prior TKI (for <= 30days): dasatinib 5; imatinib 3; nilotinib 1. Response to lower dose dasatinib is shown in Table 1.At 3 months, 93% patients had achieved early molecular response (BCR-ABL PCR <10%) to therapy. Ten patients had BCR-ABL PCR < 1% (equivalent to CCyR) at 3 months but tested positive by FISH as shown in Table 2. At 6 months, 7 of these 10 patients that were FISH positive at 3 months, achieved CCyR (FISH 0%); of these 7 patients, 6 patients achieved a MMR. Three of the remaining 10 patients that were FISH positive at 3 months, have not yet reached (NR) the 6 month follow-up.Nine patients had treatment interruption of up to 14 days in the 1st 3 months of therapy: gastrointestinal bleed 2; thrombocytopenia 3; transaminitis 2; renal dysfunction 2. None of these patients required dose modification and tolerated dasatinib once it was resumed.Seven of these 9 patients with dose interruption have achieved early response to therapy while 2 patients have not yet reached the 3 month follow-up. Therapy was discontinued in 1 patient secondary to subdural hemorrhage (traumatic), while 1 patient at 6 months was deemed a failure due to lack of cytogenetic response and BCR/ABL PCR > 10%, requiring an increase in dasatinib dose to 100mg daily. None of the patients have developed pleural effusion so far.Conclusion: Dasatinib at 50 mg daily dose is active and well-tolerated in newly diagnosed CML-CP. It should be further explored as a new potential dose-schedule option in CML. [Display omitted] DisclosuresCortes:Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Teva: Research Funding; Pfizer: Consultancy, Research Funding; Sun Pharma: Research Funding; ARIAD: Consultancy, Research Funding; BMS: Consultancy, Research Funding. Jabbour:Bristol-Myers Squibb: Consultancy. Pemmaraju:Cellectis: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Stemline: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria. Bose:Incyte Corporation: Honoraria. Thompson:Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kantarjian:Delta-Fly Pharma: Research Funding; Novartis: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding.

Activation of p53 Enhances Antileukemia Activity of Bcr-Abl Tyrosine Kinase Inhibitors in a Murine Model of CML

Although CML patients under Bcr-Abl tyrosine kinase inhibitor (TKI) therapy have experienced high response rates and long-time survival, 50% or more patients relapse upon therapy cessation, largely because TKIs do not eliminate CML stem cells. We previously reported that inhibition of antiapoptotic Bcl-2 proteins synergized with TKIs to eradicate CML stem/progenitor cells. The tumor suppressor p53 induces apoptosis in part by modulating Bcl-2 family proteins. Although rarely mutated in CML, p53 activity is antagonized by Bcr-Abl tyrosine kinase-regulated MDM2. We hypothesize that activation of p53 by MDM2 inhibition will synergize with TKIs to eliminate CML cells. Using an inducible transgenic Scl-tTa- BCR-ABL mouse model, we first determined the expression of p53 and its target genes in bone marrow (BM) cells of Tet-on (control) and Tet-off (CML) mice. We found markedly increased RNA levels of p53 and its targets Bax, Noxa, and MDM2 in CML as compared to control mice. Using CyTOF mass cytometry and cytofkid automatic subset detection/clustering based on the expression of cell surface markers, we then determined p53 and its target proteins in bulk and Lin-Sca-1+cKit+ (LSK) populations at single cell levels and found overexpression of p53 and its targets such as Noxa in CML BM cells (CD45+) compared to controls. The differential expression was most pronounced in the LSK populations. For both control and CML mice, LSK cells expressed significantly higher p53 protein than their respective bulk cell population. We next evaluated the sensitivity of these cells to BH3 peptide priming. CML BM cells and more so LSK populations were more sensitive to BH3 peptides, especially to Noxa peptide than cells from control mice (P=0.044 in CD45+ and P=0.0005 in LSK populations) suggesting that CML cells are more primed to apoptosis stimuli. Finally, we tested the antileukemia activity of combined MDM2 and Bcr-Abl inhibition in mice engrafted with BM cells from Scl-tTa- BCR-ABL /GFP mice. Combined inhibition of Bcr-Abl tyrosine kinase (imatinib) and MDM2 (DS-5272) strongly reduced BM leukemic populations and LSK cells, increased Noxa, decreased leukemia burden, and significantly prolonged survival (P=0.0021 vs. control). Median survival days for control, imatinib, DS-5272, and the combination groups were 85, 110, 155, and 248; respectively.Our results demonstrate that CML cells/stem cells have increased p53 signaling and are highly primed to undergo apoptosis. Combined p53 activation and Bcr-Abl tyrosine kinase inhibition targets CML stem/progenitor cells and has the potential to significantly improve response and cure rates of CML. DisclosuresCarter:Daiichi Sankyo: Research Funding; novartis: Research Funding. Seki:Daiichi Sankyo: Employment. Andreeff:Daiichi Sankyo: Consultancy.